Effect of Naturally Occurring E2F-4 Alterations on Transcriptional Activation and Proliferation in Transfected Cells

Abstract

SUMMARY:E2F is a family of transcription factors implicated in the regulation of gene expression required for progression through the G 1 -S transition. We have previously detected tumor-specific mutations at a trinucleotide repeat coding sequence of E2F-4 gene in a subset of human sporadic colorectal cancers. The purpose of this study was to investigate the potential functional consequences of these E2F-4 mutations. We transfected NIH3T3 fibroblasts with expression constructs containing wild-type as well as mutant E2F-4 cDNA, and the effect of the E2F-4 mutations on proliferation was examined. Alteration in transactivation of the E2F consensus promoter sequence was also examined by transient cotransfection of a E2F-4 with a DP-2 construct into cultured human cells. Transfected cell clones overexpressing mutant E2F-4 grew more rapidly and showed higher proliferative activity by increased immunohistochemical staining for proliferating cell nuclear antigen (PCNA). All three mutant forms of E2F-4 showed elevated transactivation of the E2F consensus promoter sequence. Thus, expression of mutant E2F-4s confers a growth advantage in vivo, and this effect may be related to the acquisition of a neoplastic phenotype. (Lab Invest 2001, 81:1565-1573.T he E2F family of transcription factors (E2F-1, -2, -3, -4, -5, and -6) has been implicated in the regulation of cell cycle, particularly at the level of G 1 -S progression, as well as in the regulation of apoptotic

Expression of E2F‐4 in invasive breast carcinomas is associated with poor prognosis

“…They demonstrated that the subcellular location of E2F‐4 was regulated in a cell cycle‐dependent manner. Takashima et al 23 also reported expression of E2F‐4 in both nuclei and cytoplasm. In contrast, Mady et al 20 found that E2F‐4 protein expression was strictly nuclear in position in normal and neoplastic colorectal tissues using the same antibody that we have utilized in this study.…”

“…Breast carcinoma cells showed a nuclear and/or granular cytoplasmic staining pattern (Figure 1). Because the subcellular localization of E2F‐4 is related to its functional state and its role in the cell cycle and cell growth 23, we assessed cytoplasmic and nuclear staining separately. Only staining of the invasive portion of the tumours was considered.…”

“…In vitro , overexpressed E2F‐4 is localized primarily in the cytoplasm and preferentially binds to p107/p130 22. However, endogenous E2F‐4 is predominantly nuclear in quiescent cells and, by immunostaining, overexpressed E2F‐4 is present in a nuclear location 17, 23. This nuclear localization allows E2F‐4 to have a maximal effect on regulation of the cell cycle; however, it is still unclear whether it has a functional role when localized in the cytoplasm.…”

Nuclear expression of E2F4 induces cell death via multiple pathways in normal human intestinal epithelial crypt cells but not in colon cancer cells

“…Interestingly, E2F4 gene possesses a serine repeat trinucleotide (ACG) n , which is frequently mutated in various tumors including human sporadic colorectal cancers (29). These E2F4 mutations cause elevated transactivation of E2F consensus promoter sites and confer a growth advantage in rodent fibroblasts and HEK293 cells (74). It remains unknown, however, whether these genetic alterations affect E2F4 function in normal human intestinal crypt cells, especially with regard to the regulation of cell proliferation vs. cell survival.…”

E2F4 (E2F transcription factor 4, p107/p130-binding)

“…The more frequent mutations observed are the deletion or the addition of a trinucleotide AGC and the deletion of 7 trinucleotides (Yoshitaka et al, 1996;Souza et al, 1997;Ikeda et al, 1998;Moriyama et al, 2002). Furthermore, Takashima et al, 2001, studied the impact of E2F4 mutations and observed an increase in nuclear expression, in transcriptional activity as well as in proliferation rate of fibroblasts overexpressing these mutants.…”